Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 21 December 22016 and 10 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
The purpose of this study was to assess the general systemic toxic potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of GL500, a plasticizer, by oral gavage administration for at least four weeks.
Animal Model
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHan™;WIST strain was used because of the historical control data available at this laboratory.
Route of Administration
The oral gavage route of administration was chosen to simulate the conditions of potential human exposure during manufacture, handling or use of the test item.
Rationale for Dose Level Selection
The doses used in this study (0, 100, 330 and 1000 mg/kg/day) were selected in conjunction with the Sponsor.
The dose levels were based on the results of a 2-week preliminary toxicity study in the Han Wistar rat (Envigo Study NO. LD61QW).
In the 2-week study the animals were dosed at 250, 500 or 1000 mg/kg/day. There were no deaths, the general appearance and behavior of the animals were unaffected by treatment, body weight, food consumption and water intake were also unaffected and the macroscopic examination did not reveal any findings of significance. The only change of note seen in the study was that the liver weights of males and females given 1000 mg/kg/day were slightly high after two weeks of treatment.
Based on the above findings the dose levels selected for this study were 0, 100, 330 and 1000 mg/kg/day. 330 mg/kg/day was selected in preference to 300 mg/kg/day since a dose of  300 mg/kg/day is a guidance value for Category 2 classification for specific target organ toxicity following repeated exposure (UN GHS classification system 2013).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identification: GL500
Test item (alternative names): LGflex GL500
GL500
GL520
CAS No: 1571954-81-8
Action of test item: Plasticizer
Appearance/Physical state: Clear colorless liquid
Batch: GLFG160607
Purity: 99.8%
Expiry date: 07 June 2017
Storage conditions: Room temperature in the dark
Specific details on test material used for the study:
Test item: GL500.
Test item identity (including alternative names): LGflex GL500, GL500, GL520.
CAS number: 1571954-81-8.
Intended use: Plasticizer.
Appearance: Clear colorless liquid.
Storage conditions: At ambient temperature (10 to 30C) and protected from light (although may be used and formulated in light).
Supplier: Sponsor.
Batch number: GLFG160607.
Expiry date: 1 June 2017.
Purity: 99.8%.
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 g representative sample was taken from the batch of test item, placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™;WIST ra
Sex:
male/female
Details on test animals and environmental conditions:
Animals
Strain/Species RccHan™;WIST rat.
Supplier Envigo RMS (UK) Ltd.
Number of animals ordered 44 males and 48 females.
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization Males: eight days prior to the commencement of treatment.
Females: 22 days prior to the commencement of treatment.
Age of the animals at the start of treatment Males 86 to 92 days old.
Females 100 to 106 days old.
Weight range of the animals at the start of the study Males 311 to 366 g.
Females 207 to 253 g.

Allocation and Identification
Allocation On arrival and non-selective allocation to cages.
Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study.
On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced by Study Management to ensure variations in body weight of animals did not exceed  20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.
Identification of animals Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal Replacement
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before treatment Body weight range extremes Two females
Irregular estrous cycle One female

Animal Care and Husbandry
Environmental Control
Rodent facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24ºC and 40-70%. There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage
Pre-pairing : up to five animals of one sex
Pairing : one male and one female
Males after mating : up to five animals
Gestation : one female
Lactation : one female with litter

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified pelleted diet.
A sample (100 g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30C) until finalization of the report. Samples were discarded after finalization of the report.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted (removed overnight before blood sampling for adult hematology, blood chemistry and thyroid hormone investigations. For one female, 3F 74, food was removed overnight when there was no requirement for this to occur as the animal was not required for blood sampling).
Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Test Item Preparation
Formulation
Group Treatment Dose (mg/kg/day) Formulated concentration (mg/mL) Volume dose (mL/kg)
1 Vehicle 0 0 5
2 GL500 100 20 5
3 GL500 330 66 5
4 GL500 1000 200 5

Correction factor None.
Vehicle Corn oil.
Method of preparation The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test item was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until it appeared homogeneous. A series of formulations at the required concentrations were prepared in ascending order.
Frequency of preparation Weekly.
Storage of formulation Refrigerated (2 to 8°C).
Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.
Details on mating procedure:
Mating Procedure
Pairing commenced After a minimum of two weeks of treatment.
Male/female ratio 1:1 from within the same treatment groups.
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation Analysis
Stability and homogeneity Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. Formulations were found to be homogenous and stable for 15 days when stored refrigerated (2 to 8°C) and for one day when stored at ambient temperature (15 to 25°C).
Achieved concentration Samples of each formulation prepared for administration in Weeks 1, Week 4 of treatment (males only) and on Day 12 of lactation (females only) were analyzed for achieved concentration of the test item.

Preparation of Calibration Standards
A primary standard solution (100µg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50mg) of GL500 in Acetone (50mL). A secondary standard solution (50µg/mL) was prepared by appropriate dilution of the primary standard using acetonitrile/water 90/10 v/v.
Solutions for instrument calibration were prepared by appropriate dilution of the secondary standard using acetonitrile/water 90/10 v/v and contained GL500 at nominal concentrations of 0.5µg/mL, 1.0µg/mL, 2.0µg/mL, 3.0 µg/mL 4.0 µg/mL and 5.0µg/mL.
Calibration solutions were injected onto the HPLC, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the chromatographic section.

Preparation of Test Samples
A representative sample of test formulation (1 mL, accurately weighed) and dissolved using ultrasonic vibration in a suitable volume ofacetone. The extract was diluted usingacetone and then acetonitrile/water 90/10 v/v, to provide a solution containing GL500 at an expected concentration within the range 1.0µg/mL to 4.0 µg/mL. The concentration of GL500 in the final solution was quantified by HPLC using UV detection as detailed in the chromatographic section.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (Corn oil) with known amounts of GL500.The prepared procedural recoveries were analyzed in accordance with the analytical procedure.

Instrumentation Parameters
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Poroshell 120 SB-C18, 2.7 µm, 4.6 ×100 mm
Column temperature: 45°C
Sample temperature: Ambient
Mobile Phase A: Acetonitrile/water/ortho-Phosphoric acid 90/10/0.1 v/v/v
Flow rate: 1.0 mL/min
Rinse solvent/Needle wash: Acetonitrile/water 90/10 v/v
Detector wavelength: UV, 241 nm
Injection volume: 10 µL
Run time: 12 minutes
Approximate retention time: Peak 1:2.24 min
Peak 2:3.99 min
Peak 3:8.38min

Calculations
The peak area response for GL500 in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for GL500 in sample and procedural recovery chromatograms was measured.

Validation of the Analytical Procedure
The analytical procedure was validated by determining the following parameters:
The specificity of the chromatographic analysis in control sample chromatograms.
The limit of detectionand quantificationwas estimated by examination of control vehicle chromatograms in order to calculate a test itemconcentrati on based on a peak height response equivalent to three times baseline noise.
The linearity of detector response over the calibration standard concentration range.
The repeatabilityof the lowest and highest concentration calibration standards.
The method accuracy and precision, by determining sixprocedural recoveries at nominal concentrations of1 mg/mL and 200 mg/mL during the me thod validation.

Homogeneity and Stability in VehicleFormulations
The homogeneity and stability of GL500 in corn oil formulations was assessed at nominal concentrations of1 mg/mL and200 mg/mL, during ambient and refrigerated storage. Freshly prepared specimen formulations (400 mL) were equally sub divided into 4amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Ambient Temperature Storage (15-25ºC)
On receipt, the contents of one bottle of each formulation were mixed by 20-fold inversion followed by magnetic stirring. After stirring for 20 minutes (representing 0 hour) 1, and 2hours, single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the continuously stirred formulation.
The remainder of the bottle was stored at ambient temperature and after 1 days storage,the contents were remixed and sampled as detailed above.

Refrigerated Storage (2-8ºC)
The remaining bottleswererefrigerated on receipt and on Day1, Day 8 and Day 15;the appropriate bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion followed by magnetic stirring for 20minutes and single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the stirred formulation.
Following zero hour sampling, 4 additional samples were taken (1 mL, accurately weighed) directly into volumetric flasks and were stored pending future analysis. On Day 8 and Day 15, two samples were removed from storage and analyzed according to the analytical procedure.

Concentration of Dose Formulations
For Week 1, Week 4 and Day 12 of Lactation, freshly prepared test formulations were sampled (4 × 1mL, accurately weighed) by Pharmacy personnel and submitted for analysis. Duplicate samples were analyzed in accordance with the analytical procedure, and the remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.

RESULTS
Method Validation
The analytical procedure was successfully validated for GL500 in corn oilwith respect to the specificity of chromatographic analysis, limit of detectionand quantification, linearity of detector response, repeatability, method accuracy and precision. Results are summarized below:
The specificity of the HPLC assay was demonstrated by the absence of a peak at the characteristic retention time for Test Itemin the control sample chromatogram.
The limit of detection and quantificationwas estimated as 0.332µg/mLand 1.11µg/mLrespectively.
Linearity was confirmed over the nominal concentration range 0.5 µg/mL to 5 µg/mL with a coefficient of determination >0.999;
The repeatability was <1% for six replicate injections of standard solutions containing Test Item at nominal concentrations of 0.5 µg/mL and 5 µg/mL.
Method accuracy and precision were confirmed and with a mean procedural recovery value of 97.2% (CV=0.70%, n=5) for 1mg/mL and 102.9% (CV=0.95%, n=5) for 200 mg/mL.

Homogeneity and Stability of Dose Formulations
The homogeneity and stability of GL500 in corn oilformulations was assessed with respect to the level of concentration at nominal concentrations of 1 mg/mL and 200 mg/mL.
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2hours, and on re-suspension following storage at ambient temperature for 1day and refrigeration for up to 15days. At each time-point, the mean analyzed concentration for the three samples remained within 5% of the initial time zero value and the coefficient of variationwas less than 3%. On Day 8, the high level formulation (200mg/mL) showed a low result for the top sample, which was confirmed by re-dilution of the sample. It is considered that this is a sampling error as the middle and bottom samples are within acceptable limits and the Day 15 samples are all within limits.
Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method

Concentration ofDose Formulations
The mean concentrations of GL500 in test formulations analyzed during the study were determined. The mean concentrations were within applied limits +10/-15%, confirming the accuracy of formulation. The % difference from mean values were within 2% confirming precise analysis. Recovery results were within ±7.5% of the validated mean.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detectionand quantification, linearity of detector response, repeatability, methodaccuracy and precision.
The homogeneity and stability was confirmed for GL500 in corn oilformulations at nominal concentrations of1 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2hours, ambient temperature storage for1 dayandrefrigerated storage for up to 15 days
The mean concentrations of GL500 in test formulations analyzed for the study were within +10/-15%of nominal concentrations, confirming accurate formulation.



Duration of treatment / exposure:
Duration of Treatment
Males Two weeks before pairing up to necropsy after a minimum of five weeks of treatment (animals were killed in Week 6).
Females Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation (approximately seven weeks).
Animals of the F1 generation were not dosed.
Frequency of treatment:
Daily
Details on study schedule:
The study consisted of one control and three treated groups identified as follows.
The F0 generation of the study was identified as follows:
Group Treatment Dose (mg/kg/day) Number of animals Animal numbers
Male Female Male Female
1 Control 0 10 10 21-30 61-70
2 GL500 100 10 10 11-20 51-60
3 GL500 330 10 10 31-40 71-80
4 GL500 1000 10 10 1-10 41-50

Some serial observations needed to be performed without the knowledge of the treatment group; therefore the animal numbering system was such that it was not easy to identify a treatment group from the animal number.
The F1 generation received no direct administration of the test item, GL500. Any exposure to the test item or metabolites was through the mother to the offspring in utero and/or through the milk.

Administration
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at Constant doses in mg/kg/day.
Volume dose 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Once daily at approximately the same time each day. (See Section 4 for one deviation that occurred when dosing on one day exceeded the time of dosing on the previous day by more than +2 hours).
Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Calculated from the most recently recorded scheduled body weight
Dose / conc.:
330 mg/kg bw/day (nominal)
Remarks:
Calculated from the most recently recorded scheduled body weight
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Calculated from the most recently recorded scheduled body weight
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Route of Administration
The oral gavage route of administration was chosen to simulate the conditions of potential human exposure during manufacture, handling or use of the test item.
Rationale for Dose Level Selection
The doses used in this study (0, 100, 330 and 1000 mg/kg/day) were selected in conjunction with the Sponsor.
The dose levels were based on the results of a 2-week preliminary toxicity study in the Han Wistar rat (Envigo Study NO. LD61QW).
In the 2-week study the animals were dosed at 250, 500 or 1000 mg/kg/day. There were no deaths, the general appearance and behavior of the animals were unaffected by treatment, body weight, food consumption and water intake were also unaffected and the macroscopic examination did not reveal any findings of significance. The only change of note seen in the study was that the liver weights of males and females given 1000 mg/kg/day were slightly high after two weeks of treatment.
Based on the above findings the dose levels selected for this study were 0, 100, 330 and 1000 mg/kg/day. 330 mg/kg/day was selected in preference to 300 mg/kg/day since a dose of  300 mg/kg/day is a guidance value for Category 2 classification for specific target organ toxicity following repeated exposure (UN GHS classification system 2013).

Examinations

Parental animals: Observations and examinations:
Serial Observations
Clinical and Behavioral Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males Week 1 - daily
Week 2 onwards - once each week
F0 females Week 1 - daily
Week 2 - once
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12
Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation
One to two hours after completion of dosing of all groups
As late as possible in the working day

Detailed physical examination and arena observations
Before treatment commenced, during each week of treatment, on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons; therefore observations were made on these occasions without “blinding”.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the five lowest numbered lactating females in each group at Days 7-9 of lactation. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, male cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. For females, animals were removed and returned to their home cages by an assistant, such that the observer was unaware of the treatment group. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.
The following measurements, reflexes and responses were recorded:
Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores probe/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction

Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response

Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression

Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

Motor activity
During Week 5 of treatment for males and at Days 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the five lowest numbered lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

Body Weight
The weight of animals was recorded as follows:
F0 males Weekly during acclimatization.
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.
On the day prior to necropsy (before withdrawal of food) and on the day of necropsy.
F0 females Weekly during acclimatization.
Before dosing on the day that treatment commenced (Week 0) and weekly before pairing.
Days 0, 6, 13 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day prior to necropsy (before withdrawal of food, which was Day 13 of lactation) and on the day of necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
For females after mating food consumption was performed to match the body weight recording:
Days 0-5, 6-12 and 13-19 after mating
Days 1-3 , 4-6 and 7-12 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

Parturition Observations and Gestation Length
Duration of gestation Time elapsing between the detection of mating and commencement of parturition.
Parturition observations From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Hematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food at the following occasion:
Occasion Animals
At termination The five lowest numbered surviving males and the five lowest numbered females with a surviving litter, in each dose group.

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

* Derived value calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using an ACL series analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood Chemistry
Blood samples were collected after overnight withdrawal of food at the following occasion:
Occasion Animals
At termination The five lowest numbered surviving males and the five lowest numbered females with a surviving litter, in each dose group.

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

Thyroid Hormone Analysis
Blood samples were collected as follows:
Occasion Animals
At termination All surviving F0 adult males and females
Day 4 of age F1 offspring, two females per litter (where possible) - no pups were allocated to these procedures if the resultant live litter size would drop below ten/litter.
- one for T4 (serum)#
- one for TSH (plasma)
# priority given to serum sample
Day 13 of age F1 offspring, two males and two females per litter (where possible)
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female)
# priority given to serum sample

Sequence of blood sampling on each occasion In order to minimize any potential confounding effect of the time of day of blood sampling, the order of blood sampling was controlled to allow satisfactory inter-group comparisons.
Conditions F0 animals: Following overnight deprivation of food
F1offspring: No overnight deprivation of food
Anesthetic F0 animals: Isoflurane
F1 Day 4 of age offspring: None
F1 Day 13 of age offspring: None
Blood sample site F0 adults: Sublingual vein
F1 Day 4 of age offspring: Decapitation
F1 Day 13 of age offspring: Decapitation
Anticoagulant Plasma samples: K2 EDTA
Microtainers used for collection of samples did not contain separator gel.
Serum samples: None (plain tubes without clotting activators)
Blood volume F0 animals: 2 x 0.5 mL
F1 offspring: maximum possible
Processing Plasma samples: Samples were kept on wet ice prior to centrifugation and commenced within 30 minutes of sampling.
Serum samples: Samples were kept at ambient temperature (15 to 25C) for a minimum of 60 minutes prior to centrifugation.
Centrifugation conditions At 2000g for ten minutes at 4°C.
Number of aliquots per sample All available plasma/serum transferred to appropriately labelled polypropylene “cryo” tubes using plastic disposable pipettes.
Final storage conditions Deep frozen (approximately -60 to -90ºC).
Fate of samples Dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Envigo.
Thyroid hormone analysis Performed by the Department of Bioanalysis, Envigo.
Oestrous cyclicity (parental animals):
Dry and wet smears were taken as follows:
Dry smears For 15 days before pairing using cotton swabs.
Wet smears Using pipette lavage during the following phases:
For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.

After pairing until mating.

For four days before scheduled termination (nominally Day 11 to 14 of lactation).

Litter observations:
Records Made During Littering Phase
Clinical observations Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights Days 1, 4, 7 and 13 of age.
Ano-genital distance Day 1 - all F1 offspring.
Nipple/areolae count Day 13 of age - male offspring.
Postmortem examinations (parental animals):
Terminal Investigations
Method of Kill
All adult animals Carbon dioxide asphyxiation with subsequent exsanguination.
Offspring - selected for thyroid hormone sampling on Day 4 or 13 of age Decapitation.
Offspring - all other Intraperitoneal injection of sodium pentobarbitone
Sequence To allow satisfactory inter-group comparison.

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Time of Necropsy
F0 males After Week 5 investigations completed.
F0 females failing to produce a viable litter Day 25 after mating.
F0 females Day 14 of lactation (following terminal blood sampling)
F1 offspring Selected offspring for thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.
The organs weighed, tissue samples fixed and sections examined microscopically are detailed in Table 1, below.
Postmortem examinations (offspring):
Pathology procedures for F1 offspring on Day 13 of age, premature decedents and grossly abnormal offspring culled on Day 4 of age
Necropsy
Tissue and regions examined Weigh Fix
Abnormalities *
Thyroids *
* Organs weighed, samples fixed or sections examined microscopically

Offspring
Premature deaths Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
In addition, where possible, thyroid gland tissue was preserved.
F1 offspring on Day 4 of age Blood sampling required (See Section 3.6.10).
Externally normal offspring discarded without examination.
Externally abnormal offspring examined, and tissues retained pending possible future examination.
F1 offspring on Day 13 of age Blood sampling required (See Section 3.6.10).
All animals (but not including those selected for thyroid hormone analysis) were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Thyroid glands were preserved from one male and one female in each litter.
Animals selected for thyroid hormone analysis: externally normal offspring discarded without examination; externally abnormal offspring examined.
Statistics:
See "Any other Information", below

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No signs were seen in association with the dosing procedure in males or females. There were no signs seen during the weekly detailed physical examinations that were considered to be treatment related.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains for males receiving GL500 at 1000 mg/kg/day were low, when compared with the controls, during the first two weeks of treatment, were similar to controls from Week 2 to 4 but a mean body weight loss was recorded in Week 4 to 5. These changes resulted in a low overall body weight gain for these animals (60 % of control).
Slight group mean body weight losses were seen in males receiving 100 or 330 mg/kg/day in Week 4 to 5 and the overall gain for males receiving 330 mg/kg/day was 88% of the controls. The overall body weight gain for males receiving 100 mg/kg/day was similar to controls.
Overall body weight and body weight change for females prior to pairing were similar to controls for animals receiving 100 or 1000 mg/kg/day, however, females receiving 330 mg/kg/day had a slight group mean body weight loss in the first week followed by a small weight gain in Week 2.
During gestation the body weight and body weight gains of females receiving 100, 330 or 1000 mg/kg/day were similar to controls from Day 0 to 13 but were lower than controls from Days 13 to 20 (93, 85 and 72 % of control for females receiving 100, 330 or 1000 mg/kg/day, respectively). This resulted in low overall gestational weight gains in females receiving 330 or 1000 mg/kg/day (91 and 82% of control).
During the lactation phase of this study there was no effect on body weight.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males receiving GL500 at 1000 mg/kg/day ate slightly less food in all weeks of treatment, when compared with the controls (approximately 94% of controls).
There was no effect of treatment on food consumption in females prior to pairing, during gestation or during lactation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
For the males, the haematological investigation in Week 6 did not identify any toxicologically significant differences from controls. However, the investigation performed for females on Day 14 of lactation revealed the following treatment-related findings:
Eosinophil counts were statistically significantly low for females receiving GL500 at 100, 330 or 1000 mg/kg/day (45% of control for all groups).

Large unstained cell counts were high for females receiving GL500 at 1000 mg/kg/day, (400% of control); statistical significance was not attained.

Activated partial thromboplastin time was slightly reduced for females receiving GL500 at 1000 mg/kg/day (87 %); statistical significance was not attained. The group mean value was influenced by two animals, 4F 43 and 4F 49 (with clotting times of 10.7 and 10.8 seconds, respectively. The lowest value in the control group was 12.9 seconds).

In addition, hematocrit and red blood cell counts were slightly high in all groups of females receiving GL500 (statistical significance attained), however, the differences from control were small and there was no relationship to dose. These changes were therefore attributed to normal biological variation.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood chemistry investigations, in Week 6 of treatment for males and on Day 14 of lactation for females, revealed the following treatment-related findings, when compared with the controls:
Alkaline phosphatase activities were high in females receiving 1000 mg/kg/day (142% of control); statistical significance attained. The group mean was influenced by two animals, 4F 41 and 4F 49 (with results of 99 and 125 U/L, respectively. The highest value in the control group was 69 U/L).

Bile acid concentrations were high in males and females receiving 1000 mg/kg/day (202 and 313% of control for males and females, respectively). For females, the group mean value was influenced by two animals, 4F 43 and 4F 49 (with results of 160.5 and 135 mmol/L, respectively. The highest value in the control group was 41.5 mmol/L).

Creatinine and triglyceride concentrations were slightly high in males (115 and 136 %, respectively) and females (112 and 173 %, respectively) receiving 1000 mg/kg/day; statistical significance was only attained for triglycerides in males.
Plasma chloride concentrations were slightly high in two females receiving 1000 mg/kg/day (both animals had values of 102 mmol/L). Statistical significance was not attained.

All other differences from controls were minor and were attributed to normal biological variation.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Detailed Physical Examination and Arena Observations

There were no signs seen during the weekly detailed physical examinations that were considered to be treatment related.
At the weekly examination on Day 14 of gestation, animal 2F 51 (100 mg/kg/day) had dark red discharge from the vagina. The general condition of the animal was satisfactory and she subsequently gave birth to a live litter.

Sensory Reactivity Observations and Grip Strength
Sensory reactivity observations and grip strength were similar for animals treated at 100, 330 or 1000 mg/kg/day, when compared with the Controls.

Motor Activity
Motor activity, assessed by low and high beam breaks over a 60 minute period, was unaffected by treatment with GL500 in males or females.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The evaluation of organ weights of males after 6 weeks of treatment and of females on Day 14 of lactation revealed effects in animals given 1000 mg/kg/day. These comprised: slightly high adjusted liver weights in males and females (116% of controls for both sexes), with the difference attaining statistical significance; low unadjusted (absolute) ovary weights in females (85 % of control) and slightly low, but not statistically significant, adjusted thymus weights in males and females (87 % of control).
In addition, males given 1000 mg/kg/day had slightly low body weight adjusted levator ani bulbocavernosus muscle complex, prostate and seminal vesicle weights (90%, 90% and 87% of controls, respectively), however, these differences did not attain statistical significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination performed after 5 weeks of treatment (males) or on Day 14 of lactation (females) revealed the following changes in the kidneys.
Kidneys
At the macroscopic examination on Day 14 of lactation, pale kidneys were seen in two females given 1000 mg/kg/day and one female given 330 mg/kg/day.
A mass was seen in the ovary of one female (Animal No. 56) given 100 mg/kg/day.
The incidence and distribution of all other findings were considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment Related Findings
Changes related to treatment with GL500 were seen in the kidneys and urinary bladder.
Kidneys
An increased incidence of cortical tubular hyaline droplets was seen in males given 1000 mg/kg/day. Cortical tubular basophilia was seen in rats given 330 or 1000 mg/kg/day. Cortical tubular necrosis/degeneration and/or cortical tubular dilatation were seen in females given 330 or 1000 mg/kg/day.

Summary of treatment related findings in the kidneys
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 330 1000 0 100 330 1000
Cortical tubular basophilia
Minimal 0 0 1 1 0 0 2 3
Total 0 0 1 1 0 0 2 3
Cortical tubules with hyaline droplets
Minimal 1 0 1 3 0 0 0 0
Slight 0 0 0 1 0 0 0 0
Total 1 0 1 4 0 0 0 0
Cortical tubular necrosis/ degeneration
Minimal 0 0 0 0 0 0 1 2
Total 0 0 0 0 0 0 1 2
Cortical tubular dilatation
Minimal 0 0 0 0 0 0 2 1
Total 0 0 0 0 0 0 2 1
No. of tissues examined 5 5 5 6 5 6 5 5

Urinary bladder
Transitional cell hyperplasia was seen in females given 1000 mg/kg/day. In addition, a case of mucosal ulceration with associated transitional cell hyperplasia was also seen in one female given the same dose.

Summary of treatment related findings in the urinary bladder
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 330 1000 0 100 330 1000
Transitional cell hyperplasia
Minimal 0 0 0 0 0 0 0 2
Slight 0 0 0 0 0 0 0 2
Total 0 0 0 0 0 0 0 4
Ulceration
Moderate 0 0 0 0 0 0 0 1
Total 0 0 0 0 0 0 0 1
No. of tissues examined 5 5 5 5 5 5 5 6
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males.

Mean serum T4 concentrations (pg/mL)
Group Treatment Dose (mg/kg/day) F0 Males
1 Control 0 Mean 40100
SD 6450
CV 16.1
N 10
2
GL500 100 Mean 45300
SD 5970
CV 13.2
N 10
3 GL500 330 Mean 48200
SD 6940
CV 14.4
N 10
4 GL500 1000 Mean 42500
SD 7420
CV 17.5
N 10

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous Cycles, Pre-Coital Interval, Mating Performance, Fertility and Gestation Length
All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period.
Estrous cyclicity, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment.
All females were not cycling before termination (Days 11-14 of lactation) and were in diestrous at termination, with the exception of one female treated at 100 mg/kg/day that was found to be in estrous prior to termination.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Litter Size, Sex Ratio and Survival Indices

When compared with the controls, the mean number of implantations in females treated at 330 or 1000 mg/kg/day was slightly low (84 and 86 %, respectively) and this resulted in a lower total litter size on Day 1 in these groups.
There was no effect of treatment on survival indices or the sex ratio of the offspring

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity and reproductive/developmental toxicity

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
330 mg/kg bw/day (nominal)
System:
other: Renal system
Organ:
kidney
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs related to parental treatment
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
On Day 1 of age the body weights of male and female offspring in all the GL500 treated groups were essentially similar to the controls and considered unaffected by parental treatment.
Thereafter, the body weights of male and female offspring in the 100 or 330 mg/kg/day group remained similar to the controls but in the 1000 mg/kg/day group the male and female offspring body weights were slightly low from Day 4 to 13 of age. This resulted in the mean overall body weight gains (Day 1 to 13) of male and female offspring in the 1000 mg/kg/day group being slightly low when compared with the controls.
Description (incidence and severity):
Thyroid hormone analysis
There was no effect of treatment on the circulating levels of thyroxine (T4) in offspring on Day 13 of age.
Mean serum T4 concentrations (pg/mL)
Group Treatment Dose(mg/kg/day) F1 female offspring on Day 13 of age F1 male offspring on Day 13 of age
1 Control 0 Mean 46700 43500
SD 7690 8110
CV 16.5 18.6
N 10 10
2
GL500 100 Mean 49200 43600
SD 8850 7230
CV 18.0 16.6
N 10 9
3 GL500 330 Mean 48100 42500
SD 6320 7980
CV 13.1 18.8
N 9 10
4 GL500 1000 Mean 43700 44100
SD 4340 7180
CV 9.9 16.3
N 8 9
Gross pathological findings:
no effects observed
Description (incidence and severity):
Offspring Macropathology
There were no signs in the decedent offspring, or offspring at termination on Day 13 of age that were considered related to parental treatment
Other effects:
no effects observed
Description (incidence and severity):
Offspring Ano-Genital Distance
Ano-genital distance of both male and female offspring on Day 1 of age showed no adverse effects of parental treatment

Offspring Nipple Count
One male offspring was observed with nipples on Day 13 of age (Group 3, Litter 78, male 1) although this was not confirmed at necropsy. No nipples were observed in all other male offspring.

Effect levels (F1)

Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
333 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
330 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Formulation Analysis

Homogeneity and stability

The homogeneity and stability of GL500 in corn oil formulations was assessed with respect to

the level of concentration at nominal concentrations of Low nominal concentration (1 mg/mL) and High nominal concentration (200 mg/mL). Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re‑suspension following storage at ambient temperature for 1 day and at refrigerated temperature for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 5% of the initial time zero value and the coefficient of variation was less than 3%. On Day 8, the high level formulation (200 mg/mL) showed a low result for the top sample, which was confirmed by re-dilution of the sample. It was considered that this was a sampling error as the middle and bottom samples were within acceptable limits and the Day 15 samples were all within limits. Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method.

 

Achieved concentrations of dose formulations

The mean concentrations of GL500 in test formulations analyzed during the study (samples taken in Week 1, Week 4 and for Day 12 of lactation) were within applied limits +10/-15%, confirming the accuracy of formulation. The percentage differences from the mean values were within 2%, confirming precise analysis. Recovery results were within ±7.5% of the validated mean.

Applicant's summary and conclusion

Conclusions:
In conclusion, oral administration of GL500 to Han Wistar rats at dose levels of 100, 330 or 1000 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 14 of lactation in females was generally well-tolerated in the adult animals but did elicit a reduction in body weight gain in animals receiving 330 or 1000 mg/kg/day.
The kidney was identified as the primary target organ with adverse effects reported in males and females given 330 or 1000 mg/kg/day (cortical tubular basophilia in males and females and cortical tubular necrosis/degeneration and/or cortical tubular dilatation in females). In the females these changes were accompanied by findings in the urinary bladder at 1000 mg/kg/day; transitional cell hyperplasia reported in four females and in one of these females mucosal ulceration was also seen.
Reproductive performance, fertility and offspring survival were unaffected by parental treatment but the mean number of implantations in females treated at 330 or 1000 mg/kg/day was slightly low and this resulted in a lower total litter size on Day 1 in these groups. From Day 4 of age growth was slightly impaired for offspring in the group receiving 1000 mg/kg/day.
In the context of this study, GL500 showed no evidence of being an endocrine disruptor.
The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 100 mg/kg/day.
Executive summary:

The purpose of this study was to assess the general systemic toxic potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of GL500, a plasticizer, byoral gavageadministration forat least four weeks.

Three groups of ten male and ten female rats received GL500 at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as the treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis (T4), estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

Results

F0 responses

There were no signs seen during the weekly detailed physical examinations that were considered to be treatment related and no signs were seen in association with dosing. Sensory reactivity, grip strength and motor activity were unaffected by treatment.

There was no effect of treatment on body weight gains in males receiving 100 mg/kg/day.  Among males receiving 330 mg/kg/day low mean body weight loss was recorded in Week 4 to 5 resulting in a low overall mean weight gain (88% of control). Males receiving 1000 mg/kg/day had low weight gains during the first two weeks of treatment and a mean body weight loss was recorded in Week 4 to 5, resulting in a low overall body weight gain (60 % of control). Body weight performance in females prior to pairing was variable with satisfactory gains recorded in females receiving 100 or 1000 mg/kg/day but inferior gains recorded for females receiving 330 mg/kg/day. During gestation the body weight and body weight gains of all groups of females receiving GL500 were similar to controls from Day 0 to 13 but were lower than controls from Days 13 to 20 (93, 85 and 72 % of control for females receiving 100, 330 or 1000 mg/kg/day, respectively). This resulted in low overall gestational weight gains in females receiving 330 or 1000 mg/kg/day (91 and 82% of control). There was no effect on body weight during the lactation phase. 

Males receiving GL500 at 1000 mg/kg/day ate slightly less food in all weeks of treatment, when compared with the controls (approximately 94% of controls). Food consumption in females was not affected by treatment.

Estrous cycles, pre-coital interval, mating performance, fertility and gestation length and index were unaffected by treatment. When compared with the controls, the mean number of implantations in females treated at 330 or 1000 mg/kg/day was slightly low (84 and 86 %, respectively) and this resulted in a lower total litter size on Day 1 in these groups.

Haematological investigations revealed no effects of treatment in males but did identify the following changes in the females: eosinophil counts were low for females treated at 100, 330 or 1000 mg/kg/day (45% of control for all groups); large unstained cell counts were high for females treated at 1000 mg/kg/day (400% of control); activated partial thromboplastin time was slightly reduced for females treated at 1000 mg/kg/day (87 %).

Blood chemistry investigations revealed the following changes which were attributed to treatment with GL500: high alkaline phosphatase activities in females treated at 1000 mg/kg/day (142% of control); high bile acid concentrations in males and females treated at 1000 mg/kg/day (202 and 313% of control for males and females, respectively); high creatinine and triglyceride concentrations in males (115 and 136 %, respectively) and females (112 and 173 %, respectively) treated at 1000 mg/kg/day; high plasma chloride concentrations in two females treated at 1000 mg/kg/day.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males.

The evaluation of organ weights for males after 6 weeks of treatment and for females on Day 14 of lactation revealed effects in animals given 1000 mg/kg/day. These comprised: slightly high adjusted liver weights in males and females (116% of controls for both sexes; low unadjusted (absolute) ovary weights in females (85 % of control) and slightly low adjusted thymus weights in males and females (87 % of control). In addition, males given 1000 mg/kg/day had slightly low body weight adjusted levator ani‑bulbocavernosus muscle complex, prostate and seminal vesicle weights (90%, 90% and 87% of controls, respectively).

Macroscopic examination of the males performed after 5 weeks of treatment revealed no test‑item related lesions. At the examination of the females, killed on Day 14 of lactation, pale kidneys were reported for one female given 330 mg/kg/day and two females given 1000 mg/kg/day.

At microscopic examination, changes related to treatment with GL500 were seen in the kidneys and urinary bladder. In the kidneys, an increased incidence of cortical tubular hyaline droplets was seen in males given 1000 mg/kg/day. Cortical tubular basophilia was seen in males and females given 330 or 1000 mg/kg/day and cortical tubular necrosis/degeneration and/or cortical tubular dilatation were seen in females given 330 or 1000 mg/kg/day. Transitional cell hyperplasia was seen in the urinary bladder of females given 1000 mg/kg/day and, in one female at this dose, mucosal ulceration with associated transitional cell hyperplasia was also reported.

F1 responses

The clinical condition of the offspring, offspring survival and sex ratio were unaffected by parental treatment.

There was no effect of parental treatment on the circulating levels of thyroxine (T4) in offspring on Day 4 or 13 of age.

Ano-genital distance of both male and female offspring on Day 1 of age and male nipple counts on Day 13 of age showed no adverse effects of parental treatment.

On Day 1 of age the body weights of male and female offspring in all the GL500 treated groups were similar to the controls. However, in the 1000 mg/kg/day group the male and female offspring body weights were slightly low from Day 4 to 13 of age, reflecting the mean overall body weight gains (Day 1 to 13) being slightly low when compared with the controls.

Macroscopic examination of offspring that died prior to scheduled termination or were killed at Day 13 of age did not reveal any findings that could be related to parental treatment.

Conclusion

In conclusion, oral administration of GL500 to Han Wistar rats at dose levels of 100, 330 or 1000 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 14 of lactation in females was generally well-tolerated in the adult animals but did elicit a reduction in body weight gain in animals receiving 330 or 1000 mg/kg/day. 

The kidney was identified as the primary target organ with adverse effects reported in males and females given 330 or 1000 mg/kg/day (cortical tubular basophilia in males and females and cortical tubular necrosis/degeneration and/or cortical tubular dilatation in females). In the females these changes were accompanied by findings in the urinary bladder at 1000 mg/kg/day; transitional cell hyperplasia reported in four females and in one of these females mucosal ulceration was also seen. 

Reproductive performance, fertility and offspring survival were unaffected by parental treatment but the mean number of implantations in females treated at 330 or 1000 mg/kg/day was slightly low and this resulted in a lower total litter size on Day 1 in these groups. From Day 4 of age growth was slightly impaired for offspring in the group receiving 1000 mg/kg/day.

In the context of this study, GL500 showed no evidence of being an endocrine disruptor.

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 100 mg/kg/day.